This project applies biochemical and molecular biological approaches to the study of the complex interplay of the molecular signaling events that occur within T cells in response to engagement of the T cell antigen receptor (TCR) and co-stimulatory receptors. It is anticipated that a better understanding of the events that occur in the normal course of T cell activation will provide logical targets to probe with regard to their importance in immune pathogenicities, such as those characterized by immunosenescence in aged animals. The current focus is upon the kinases and phosphatases that regulate the phosphorylation status of key proteinaceous and phosphoinositide-based signaling molecules. Of particular interest has been the exploration of alternative signaling pathways downstream of the TCR that may be associated with the establishment or maintenance of peripheral tolerance (anergy). In many respects the T cells from aged animals have an anergic phenotype. Previous studies have suggested that a key determinant of whether or not TCR stimulation leads to T cell activation rather than to anergy is the status of ZAP-70 activation. Furthermore, the establishment of anergy is an active signaling process. We therefore, set out to identify which signaling pathways might be involved in signaling downstream of the TCR in the absence of ZAP-70. By comparing the responses of T cell lines that differ only in their expression of ZAP-70, we found that TCR engagement was able to signal to two key intracellular pathways: the Ras/Raf/Erk pathway and the Ca2+ mobilization pathway. It was notable that while these pathways did indeed become activated, their activation kinetics were markedly different in the ZAP-70-negative cells as compared to the ZAP-70 replete cells. In addition, the activation of these pathways in the ZAP-70-negative cells was independent of phosphorylation of the transmembrane adapter protein LAT and required activation of protein kinase C, further distinguishing the TCR signaling pathways in ZAP-70-deficient and ZAP-70-replete T cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation and Ca2+ mobilization in Jurkat T cells: a ZAP-70-independent pathway requiring PKC, and a ZAP-70-dependent pathway that is PKC-independent. It seems likely that the PKC-dependent, ZAP-70-independent activation of the Erk and Ca2+ mobilization pathways is an important component of the signaling pathway leading to anergy and that it is, in part, the difference in the kinetics of activation of these pathways that determines whether TCR engagement is stimulatory or tolerizing. Additional studies related to this project have investigated the role of the lipid phosphatase PTEN in regulating growth, proliferation and activation of T cells. This phosphatase acts in opposition to the lipid kinase, PI3K. Previously our laboratory demonstrated that unlike normal T cells, the leukemic Jurkat T cell line fails to express PTEN. To explore the importance of this phosphatase in T cell function and response to TCR stimulation we have created a Jurkat T cell line that expresses PTEN under a tightly-controlled expression system. Notably, Jurkat T cells expressing PTEN seem to have little increase in susceptibility to apoptosis, but rather are smaller in size and proliferate more slowly than PTEN-negative cells. We propose from these studies that the net balance of activity between PTEN and PI3K plays a pivotal role in the progression from quiescence to the profound replication rate associated with T cell activation. Unregulated changes in this balance, as characterized by the loss of PTEN expression likely predispose towards the development of leukemia. Whether or not more subtle perturbations of the pathways regulated by these enzymes are involved in the reduced proliferative responsiveness of immunosenescent T cells remains to be established.